Methods and compositions for the detection of microbial contaminants

ABSTRACT

The invention provides methods and compositions for the detection and/or quantification of a microbial contaminant, for example, a bacterial endotoxin or a glucan, in a sample. In particular, the invention provides a test cartridge useful in the practice of hemocyte lysate-based assays for the detection and/or quantification of a microbial contaminant in a sample. In addition, the invention provides methods of making and using such cartridges. In addition, the invention provides a rapid, sensitive, multi-step kinetic hemocyte lysate-based assay for the detection and/or quantification of a microbial contaminant in a sample. In addition, the invention provides a glucan-specific lysate that can be used in a variety of assay formats, including, for example, a test cartridge, optionally configured to perform a kinetic assay.

RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Ser.No. 60/455,632, filed Mar. 17, 2003, the contents of which areincorporated by reference herein.

FIELD OF THE INVENTION

[0002] The present invention relates generally to methods andcompositions for detecting and/or quantifying microbial contaminants ina sample. More particularly, the invention relates to methods andcompositions using a hemocyte lysate for detecting and/or quantifyingmicrobial contamination in a sample.

BACKGROUND OF THE INVENTION

[0003] Microbial contamination by, for example, Gram positive bacteria,Gram negative bacteria, yeast, fungi, and molds may cause severe illnessand, in some cases, even death in humans. Manufacturers in certainindustries, for example, the pharmaceutical, medical device, and foodindustries, must meet exacting standards to verify that their productsdo not contain levels of microbial contaminants that would otherwisecompromise the health of the recipient. These industries requirefrequent, accurate, and sensitive testing for the presence of suchmicrobial contaminants to meet certain standards, for example, standardsimposed by the United States Food and Drug Administration (USFDA) orEnvironmental Protection Agency. By way of example, the USFDA requirescertain manufacturers of pharmaceuticals and invasive medical devices toestablish that their products are free of detectable levels of Gramnegative bacterial endotoxin.

[0004] Furthermore, when people become infected with Gram negativebacteria, the bacteria may produce and secrete fever-inducing bacterialendotoxins. Bacterial endotoxins can be dangerous and even deadly tohumans. Symptoms of infection may range from fever, in mild cases, todeath. In order to promptly initiate proper medical treatment, itusually is important to identify, as early as possible, the presence ofan endotoxin and, if possible, the concentration of the endotoxin in thepatient.

[0005] To date, a variety of assays have been developed to detect thepresence and/or amount of a microbial contaminant in a test sample. Onefamily of assays use hemocyte lysates prepared from the hemolymph ofcrustaceans, for example, horseshoe crabs. These assays typicallyexploit, in one way or another, a clotting cascade that occurs when thehemocyte lysate is exposed to a microbial contaminant. A currentlypreferred hemocyte lysate is amebocyte lysate (AL) produced from thehemolymph of a horseshoe crab, for example, Limulus polyphemus,Tachypleus gigas, Tachypleus tridentatus, and Carcinoscorpiusrotundicauda. Amebocyte lysates produced from the hemolymph of Limulus,Tachypleus, and Carcinoscorpius species are referred to as Limulusamebocyte lysate (LAL), Tachypleus amebocyte lysate (TAL), andCarcinoscorpius amebocyte lysate (CAL), respectively.

[0006] Routine assays that use LAL include, for example, gel clotassays, end point turbidometric assays, kinetic turbidometric assays,and endpoint chromogenic assays (Prior (1990) “Clinical Applications ofthe Limulus Amebocyte Lysate Test” CRC PRESS 28-34). These assays,however, suffer from one or more disadvantages including reagentexpense, assay speed and limited sensitivity ranges. Also, these assaystypically require that samples be sent to a testing facility removedfrom the origin of the sample being tested. As a result, it may takehours to weeks before a problem can be detected and remedied.Accordingly, there is an ongoing need for faster and more sensitivemethods, and portable test systems employing such methods, that overcomethe need to submit samples to an off-site testing facility.

SUMMARY OF THE INVENTION

[0007] The invention is based, in part, upon the discovery that it ispossible to make and use optical cartridges containing an immobilizedhemocyte lysate for use in hemocyte lysate-based assays. Thesecartridges may be used alone or in combination with optical detectors,for example, hand held optical detectors, to permit the assay of samplesin the field, thereby obviating the need to send samples to an off-sitetesting facility. Accordingly, the cartridges can be used in apoint-of-use test system. In addition, the invention is based, in part,upon the discovery of a rapid, sensitive, multi-step kinetic assay fordetermining the presence and/or amount of microbial contaminant in asample of interest. This type of assay can be implemented in acartridge, or in any other desirable assay format. In addition, theinvention is based, in part, upon the discovery of a glucan-specifichemocyte lysate and a method of making such a lysate. Theglucan-specific lysate may be incorporated into such a cartridge and/orused in a multi-step kinetic assay.

[0008] In one aspect, the invention provides a test cartridge fordetermining the presence and/or amount of a microbial contaminant in asample. The cartridge comprises a housing defining at least one fluidinlet port, at least one optical cell, and at least one conduit having afluid contacting surface that connects and thus provides fluid flowcommunication between the fluid inlet port and the optical cell. Thecartridge further comprises a hemocyte lysate disposed on a first regionof the fluid contacting surface of the conduit, so that when a sample isapplied to the fluid inlet port, the sample traverses the region andsolubilizes the hemocyte lysate during transport of the sample-lysatemixture to the optical cell. This type of cartridge can be used toperform, for example, endpoint turbidometric and kinetic turbidometricassays.

[0009] The cartridge optionally may further comprise a chromogenicsubstrate that acts as a substrate for one or more of the enzymes in thehemocyte lysate. The chromogenic substrate may be disposed in the firstregion, for example, combined with the hemocyte lysate. In this format,the sample resolubilizes or starts to resolubilize the hemocyte lysateand chromogenic substrate at substantially the same time. This type ofcartridge can be used to perform, for example, a kinetic chromogenicassay. Alternatively, the chromogenic substrate may be disposed on asecond region of the fluid contacting surface of the conduit, so thatwhen the sample moves along the conduit toward the optical cell itcontacts and reconstitutes the hemocyte lysate and chromogenic substrateat different regions of the conduit. In one embodiment, the secondregion is located downstream of the first region (i.e., the secondregion is located closer to the optical cell than the first region).This type of cartridge can be used to perform, for example, endpointchromogenic and multi-step kinetic chromogenic assays, as discussed inmore detail herein below.

[0010] In addition, a pre-selected amount of an agent representative ofa microbial contaminant, or “spike”, such as a bacterial endotoxin, a(1→3)-B-D glucan, or other microbial cell wall constituent, is disposedon a region of the fluid contacting surface of the conduit. Theinclusion of the agent or spike is particularly useful as it provides apositive control to demonstrate that an assay is working, and can alsodemonstrate whether an inhibitor or enhancer is present in the sample.The agent or spike may be disposed on the first region, the secondregion, or on another region of the conduit.

[0011] It is contemplated that the number of fluid inlet ports, opticalcells, and conduits in a particular cartridge may vary depending on thenumber of samples or microbial contaminants being tested at a particulartime. A cartridge may be used to simultaneously assay duplicates of thesame sample or simultaneously assay two or more different samples ofinterest. Alternatively, two or more different hemocyte lysates may bedisposed on the cartridge so that it is possible to determine thepresence and/or amount of two or more different microbial contaminantsat substantially the same time. In addition, several chromogenicsubstrates with different enzyme specificities and optical properties,for example, light absorption transmission, and/or fluorescentproperties, may be applied to the cartridge. This may permit thedetection of two or more different microbial contaminants atsubstantially the same time.

[0012] In one embodiment, the cartridge comprises a housing that defines(i) a first fluid inlet port, a first optical cell, and a first conduithaving a fluid contacting surface that connects and thus provides fluidflow communication between the first fluid inlet port and the firstoptical cell, and (ii) a second fluid inlet port, a second optical cell,and a second conduit having a fluid contacting surface that connects andthus provides fluid flow communication between the second fluid inletport and the second optical cell. Within the housing, a first hemocytelysate is disposed on a first region of the fluid contacting surface ofthe first conduit, so that when a sample is applied to the first fluidinlet port, the sample traverses the region and reconstitutes and/orsolubilizes the first hemocyte lysate during transport to the firstoptical cell. Also within the housing, a second hemocyte lysate isdisposed on a first region of the fluid contacting surface of the secondconduit, so that when a sample is applied to the second fluid inletport, the sample traverses the region and reconstitutes and/orsolubilizes the second hemocyte lysate during transport to the secondoptical cell.

[0013] In one embodiment, a chromogenic substrate is disposed on asecond region of the fluid contacting surface of the first conduitand/or a chromogenic substrate is disposed on a second region of thefluid contacting surface of the second conduit. In each embodiment, thesecond region preferably is located downstream of the first region ineach conduit. In another embodiment, different chromogenic substratesmay be disposed on fluid contacting surfaces of the first and secondconduits so that two different reactions may be monitored in the samecartridge.

[0014] In another embodiment, a pre-selected amount of an agentrepresentative of a microbial contaminant or spike is disposed on thefluid contacting surface of the first or second conduit. The spike maybe disposed on the first region or on another region of the conduit. Thespike may be useful as a positive control for the assay (i.e., indicateswhether a valid test was run), and may also provide information onwhether an enhancer or inhibitor is present in the sample.

[0015] As will be discussed in more detail below, the cartridges of theinvention may be adapted for use in a variety of different assays. Thepresence of a microbial contaminant may be indicative of, for example,past or present bacterial, yeast, fungal, or mold infection. It iscontemplated that, by using the appropriate hemocyte lysate, thecartridge may be used to detect the presence of a bacterial, yeast,fungal, or mold contaminant in a sample. The cartridges of the inventionare particularly useful at detecting the presence, and/or determiningthe amount, of a Gram negative bacterial endotoxin or glucan in asample.

[0016] During use, a sample to be tested is introduced into the sampleinlet port of the cartridge and is allowed to move to the optical cell.Movement of the sample along the conduit can be passive or can beinduced by an external force, for example, via positive or negativepressure in the conduit. For example, the sample can be pulled along theconduit to the optical cell via suction induced by a pump connected to apump port located downstream of the optical cell. A change in an opticalproperty of the sample is detected in the optical cell, the change beingindicative of the presence of a microbial contaminant in the sample. Inaddition, the time in which a pre-selected change occurs in an opticalproperty of the sample can be determined and compared against apredetermined standard curve to determine the concentration of themicrobial contaminant in the sample. The optical property may include acolor change, a change in absorbance or transmittance, a change inturbidity, a change in fluorescence or other change that can be detectedin a detector, spectrophotometer or the like. The change in the opticalproperty may be, for example, an increase in absorbance of light of apre-selected wavelength, or may be a decrease in transmission of lightof a pre-selected wavelength. As discussed below, the cartridge may beadapted to perform any one of a number of endpoint or kineticchromogenic, or turbidometric assays.

[0017] In another aspect, the invention provides methods of preparingthe cartridge by disposing, for example, by drying, a hemocyte lysateonto a solid surface of at least one conduit of the cartridge. Thehemocyte lysate may then be reconstituted into an active form uponresolubilization of the hemocyte lysate. A volume of a mixturecomprising a hemocyte lysate of interest and a resolubilizing agentand/or an anti-flaking agent is applied to the surface of at least oneconduit and dried. The hemocyte lysate used will depend upon the assayfor which the cartridge will be used. The resolubilizing agent is anagent that, either alone or in combination with another resolubilizingagent, facilitates the resolubilization of one or more components of thehemocyte lysate once the lysate is exposed to a fluid sample. Theresolubilizing agent preferably also stabilizes the lysate in its driedform. The resolubilizing agent provided in the mixture facilitates thestability of the reagents and their dissolution during the assay.Resolubilizing agents include, for example, one or more sugars, salts,or combinations thereof. Preferred sugar resolubilizing agents include,for example, mannitol, mannose, sorbitol, trehalose, maltose, dextrose,sucrose, and other monosaccharides or disaccharides. The anti-flakingagent included in the mixture further stabilizes the reagents andreduces flaking of the dried lysate. The anti-flaking agent preferablyalso stabilizes the lysate in its dried form. Preferred anti-flakingagents include, for example, one or more polymers, for example,polyethylene glycol, polyvinyl pyrolidone, polyvinyl alcohol, mannitol,dextran, and proteins, for example, serum albumin.

[0018] In one embodiment, the lysate/resolubilizing agent/anti-flakingagent mixture is disposed in a first region of at least one conduit ofthe cartridge. The mixture then is dried onto a surface of the conduitin an environment having a temperature of about 4° C. to about 40° C.,more preferably, from about 10° C. to about 35° C., more preferably,from about 15° C. to about 30° C. and a relative humidity of about 0% toabout 30%, more preferably, from about 2% to about 20%, more preferably,from about 4% to about 10%. Preferably, the temperature is about 25° C.and the relative humidity is about 5%. Drying preferably occurs forabout 10 minutes to about 8 hours, more preferably for about 1 hour in atemperature regulated drying chamber. The lysate/resolubilizingagent/anti-flaking agent mixture optionally may also include an agentrepresentative of a microbial contamination (i.e., a spike), forexample, a bacterial endotoxin, a (1→3)-B-D glucan or other microbialcell wall constituents.

[0019] In another embodiment, the mixture is dried onto the surface ofthe conduit by lyophilization or freeze drying, for example, attemperatures below 0° C., for example, from about −75° C. to about −10°C., more preferably from about −30° C. to about −20° C.

[0020] In addition, a chromogenic substrate, comprising a resolubilizingagent and an anti-flaking agent is applied to a second region of thecartridge and dried onto the cartridge as described above. Thechromogenic substrate may comprise an anti-frothing agent, for example,polyvinyl alcohol and polypropylene glycol.

[0021] Although the drying procedure is discussed in relation to a testcartridge, the drying procedure can be used to dry the lysate onto avariety of different solid supports. For example, the method can also beused to dry the lysate on the surface of a well disposed within ordefined by a solid support, for example, a 12-well or a 96-well plate.

[0022] In another aspect, the invention provides an improved methodreferred to as a two-step or multi-step kinetic assay for detecting thepresence and/or quantifying the amount of a particular microbialcontaminant in a sample. The sample to be tested is contacted with ahemocyte lysate comprising an activatable enzyme, for example, apro-clotting enzyme or a clotting enzyme, that is activated if themicrobial contaminant is present in the sample. After contacting thesample with the lysate, the sample-lysate mixture is incubated for apreselected period of time. Then, the sample-lysate mixture is contactedwith a substrate, for example, a chromogenic substrate, for theactivated enzyme. If the sample-lysate substrate mixture contains anactivated enzyme, the activated enzyme produces a change in thesubstrate, which in turn produces a predetermined change in an opticalproperty of the sample-lysate-substrate mixture. The time in which thepredetermined change occurs then is determined. The resulting time thenis compared to a predetermined standard curve to determine whether themicrobial contaminant is present in the sample and/or to determine theamount of microbial contaminant present in the sample. For example, theconcentration of microbial contaminant in a sample can be measured bycomparing the time required to produce the predetermined change in theoptical property against a predetermined standard curve of the microbialcontaminant. Using this type of assay, it is possible to adjust thetiming of the steps to produce an assay of predetermined sensitivity andduration.

[0023] The optical property measured can be a change (e.g., an increaseor decrease) in an optical property, for example, absorbance at aparticular wavelength, transmittance at a particular wavelength,fluorescence at a particular wavelength, or optical density. Forexample, the optical property may be a change in absorbance ortransmittance at a wavelength in the range from about 200 nm to about700 nm, and more preferably in the range from about 350 nm to about 450nm.

[0024] The chromogenic substrate may be any substrate for a lysateenzyme that is activated (e.g., hydrolyzed) to cause a detectablechromogenic or fluorogenic change, for example, by release of achromophore or a fluorophore, that is detectable by an optical detector.In one embodiment, the chromogenic substrate for LAL contains apara-nitroaniline chromophore, such as that, for example, in thechromogenic substrate acetate-Ile-Glu-Ala-Arg-pNA. The proteases in theLAL cleave colorless tetrapeptide to release the pNA group, which causesa color change. Cleavage of the tetrapeptide simulates the cleavagereaction of the proteases in the LAL with coagulogen, a clottingcomponent that contains the tetrapeptide. As a result, by using thischromophore, it is possible to measure the progress of the reaction bymeasuring the change in optical density at about 395 nm. Otherchromophores may include dinitrophenyl alanine, cyclohexyl alanine andthe like. Alternatively, the substrate may contain a fluorophore, forexample, 7-amino-4-methyl coumarin, 7-amino-4-trifluoromethyl coumarin,and 4-methoxy-2-naphthalyamine. Fluorogenic substrates for LAL thatcontain N-methylcoumarin as a leaving group are available from EnzymeSystems Products, Livermore, Calif.

[0025] The multi-step kinetic assay may be performed in a variety offormats, for example, in a tube, cuvette, cartridge, well on a solidsupport (such as a 96-well multi-well plate), or other vessel suitablefor use in combination with an optical detector, for example, aspectrophotometer, fluorimeter, luminometer, or the like.

[0026] In another aspect, the invention provides a method for producingan amebocyte lysate depleted of Factor C activity. The method comprisesthe steps of: (a) providing a preparation of amebocytes; and (b) lysingthe amebocytes in the presence of at least 0.05M salt to provide anamebocyte lysate preparation depleted of Factor C activity. The methodoptionally includes the step of, after step (b), removing cellulardebris, for example, cell membranes, and then harvesting the remaininglysate. The cellular debris may be sedimented by centrifugation and theremaining supernatant harvested.

[0027] In one embodiment, the salt may comprise a monovalent cation, forexample, a sodium or potassium salt. Salts useful in the practice of theinvention include, for example, sodium chloride, potassium chloride,sodium acetate, and potassium acetate. The salt concentration can be inthe range from 0.15 M to about 6 M, more preferably from about 0.25 M toabout 4 M, and more preferably from about 1 M to 2 M. However, theprecise concentration of salt necessary to remove or reduce Factor Cactivity may be determined by routine experimentation. For example,amebocytes can be lysed in the presence of difference concentrations ofsalt, and the residual lysates can then be checked to see whether acoagulin clot forms in the presence of a bacterial endotoxin. Theforegoing method can produce a glucan-specific amebocyte lysate that issubstantially free of Factor C activity. The lysate, therefore, retainsFactor G activity but is depleted of Factor C activity.

[0028] In another aspect, the invention provides an amebocyte lysatesubstantially free of Factor C activity, wherein the lysate comprises atleast about 0.25M salt and wherein the lysate is capable of producing acoagulin gel in the presence of glucan. The lysate may comprise, fromabout 0.25 M salt to about 6 M salt, from about 0.5 M salt to 4 M salt,and from about 1 M to about 2 M salt. In one embodiment, the saltcontains a monovalent cation, for example, a sodium ion or a potassiumion. For example, the salt may include sodium chloride, potassiumchloride, sodium acetate, potassium acetate, or a combination thereof.

[0029] The foregoing and other objects, features and advantages of thepresent invention will be made more apparent from the following drawingsand detailed description of preferred embodiments of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The objects and features of the invention may be betterunderstood by reference to the drawings described below in which,

[0031]FIG. 1 is a schematic representation of the coagulation systempresent in amebocytes;

[0032]FIGS. 2A-2D are schematic illustrations of an exemplary cartridgeof the invention in perspective view (FIG. 2A), top view (FIG. 2B), sideview (FIG. 2C), and end view (FIG. 2D);

[0033]FIG. 3A-3B are schematic illustrations of an exemplary cartridgeof the invention wherein each conduit has a separate fluid inlet port(FIG. 3A), and wherein two conduits share a single common fluid inletport (FIG. 3B);

[0034]FIGS. 4A-4B are schematic illustrations of an exemplary cartridgeof the invention wherein each conduit has its own fluid inlet port (FIG.4A), and wherein a pair of conduits share a single common fluid inletport (FIG. 4B);

[0035]FIGS. 5A-5D are schematic illustrations of an exemplary cartridgein which FIG. 5A is a view of a bottom half of an exemplary cartridge ofthe invention showing the locations of immobilized hemocyte lysate andchromogenic substrate, FIG. 5B is a view of a top half of an exemplarycartridge of the invention showing the location of an immobilized agentrepresentative of a microbial contaminant (i.e., spike), FIG. 5C is across-sectional view of the fabricated cartridge through section A-A′,and FIG. 5D is a cross-sectional view of the fabricated cartridgethrough section B-B′;

[0036]FIG. 6A-6D are schematic illustrations of two exemplarycartridges, in which FIG. 6A is a top view of a first embodiment of acartridge, FIG. 6B is a side view of the first embodiment of thecartridge pictured in FIG. 6A, FIG. 6C is a top view of a second,different embodiment of a cartridge, and FIG. 6D is a side view of thesecond embodiment of the cartridge of FIG. 6C;

[0037]FIG. 7 is a flow chart for an exemplary multi-step kineticchromogenic assay of the invention;

[0038]FIG. 8A-8B are schematic illustrations showing a cartridge incombination with an exemplary optical detector in which FIG. 8A showsthe cartridge being inserted into the detector, and FIG. 8B shows thecartridge actually inserted into the detector;

[0039]FIG. 9 is a graphical representation showing changes in lightabsorbance or optical density (full line) or light transmittance (dashedline) through an optical cell of an exemplary cartridge of the inventionduring a multi-step kinetic chromogenic assay;

[0040]FIGS. 10A-10B are graphical representations of absorbance valuesin an end point chromogenic assay where FIG. 10A shows the absorbancevalues of endotoxin standards (1.0 Endotoxin Units (EU)/mL (A1), 0.5EU/mL (A2), 0.25 EU/mL (A3), and 0.125 EU/mL (A4)) over time in anendpoint chromogenic assay performed in a cartridge of the invention,and FIG. 10B shows a standard curve generated by plotting the absorbancevalues of each concentration of endotoxin at T=780 seconds;

[0041]FIGS. 11A-11D are graphical representations of absorbance valuesfor a kinetic chromogenic assay performed in a cartridge of theinvention, where FIG. 11A shows the absorbance values of a 5.0 EU/mLendotoxin standard, FIG. 11B shows the absorbance values of a 0.5 EU/mLendotoxin standard, FIG. 11C shows the absorbance values of a 0.05 EU/mLendotoxin standard, and FIG. 11D is a standard curve generated byplotting the log of endotoxin concentration (X-axis) versus the log ofabsorbance value at onset time (Y axis);

[0042]FIG. 12 is a graphical representation of a standard curve for amulti-step kinetic chromogenic assay performed in a cartridge of theinvention, generated by plotting the log of endotoxin concentration(X-axis) versus the log of absorbance value at onset time (Y axis);

[0043]FIG. 13 is a graphical representation of a standard curve for asingle-step kinetic chromogenic assay performed in a microtiter plate,generated by plotting the log of endotoxin concentration (X-axis) versusthe log of absorbance value at onset time (Y axis);

[0044] FIGS. 14 is a graphical representation of a standard curve for anendpoint chromogenic assay performed in a microtiter plate, generated byplotting the absorbance values (Y axis) of each concentration ofendotoxin (X axis); and

[0045]FIG. 15 is a graphical representation of a standard curve for amulti-step kinetic chromogenic assay performed in a microtiter plate,generated by plotting the log of endotoxin concentration (X-axis) versusthe log of absorbance value at onset time (Y axis).

[0046] FIGS. 16A-B are graphical representations of amebocyte lysatekinetic reactions for standard LAL and glucan-specific LAL. FIG. 16Arepresents assays performed using lipopolysaccharide, and FIG. 16Brepresents assays performed using glucan.

[0047] Row A in each Figure represents standard LAL containing both theFactor C and Faction G cascades, Row B in each Figure representsglucan-specific lysate (i.e., Factor G-specific lysate) prepared bylysing amebocytes in 1M sodium chloride and Row C representsglucan-specific lysate prepared by lysing amebocytes in 2M NaCl. Columns1-5 represent several dilutions of the lipopolysaccharide (10 ng/ml, 1ng/ml, 100 pg/ml, 10 pg/ml, 0) or glucan (100 μg/ml, 10 μg/ml, 1 μg/ml,100 ng/ml, 0) added to each sample, wherein the concentration decreasesfrom column 1 to column 5;

[0048]FIG. 17 is a graphical representation of a logarithmic plot of aglucan standard curve obtained using a multi-step kinetic assay;

[0049]FIG. 18 is a graphical representation of an assay measuring thefluorescence emitted from different concentrations of a fluorogenicsubstrate Glu-Gly-Arg-AMC in a multi-step kinetic assay in the cartridgeformat;

[0050]FIG. 19 is a graphical representation showing the proportionaldisplacement of various concentrations of lipopolysaccharide-fluoresceinisothiocyanate (1 μg/ml, 100 ng/ml, 10 ng/ml, 1 ng/ml, 100 pg/ml, 10pg/ml, and control) by different dilutions of a fluorescein-labeledligand (1/50, 1/150, and 1/450).

[0051] In the drawings, which are not necessarily drawn to scale, likecharacters refer to the same or similar parts throughout the Figures.

DETAILED DESCRIPTION OF THE INVENTION

[0052] The invention provides an optical cartridge containing animmobilized hemocyte lysate for use in hemocyte-lysate based assays.These cartridges may be used alone or together with an optical detector,for example, a hand held optical detector. In addition, the inventionprovides a rapid, sensitive, broad range, multi-step assay that isuseful in determining the presence and/or amount of a microbialcontaminant in a sample. Although the cartridge and method may be usedseparately, they are particularly effective when combined together toprovide a system that can be used in the field to provide rapid testresults. This facilitates quicker elimination and/or treatment ofmicrobial contamination. In addition, the invention provides a Factor Cspecific lysate for detecting the presence and/or amount of glucan in asample. The lysate, therefore, can be used to determine the presenceand/or amount of a yeast or mold contaminant in a sample.

[0053] The Cartridge

[0054] It is contemplated that the cartridges of the invention may beformulated with one or more hemocyte lysates and used in a variety ofassays to detect the presence and/or amount of a microbial contaminantin a sample. A number of hemocyte lysate-based assays for the detectionand/or quantification of a microbial contaminant can be performed in thecartridge of the invention, for example, as illustrated in FIG. 2. Thecartridge may be used on its own and the test result detected by eye ormay be used in combination with an optical detector, for example, ahand-held optical detector as shown and described in U.S. Pat. No. Des.390,661.

[0055] By way of example and as illustrated in FIGS. 2A-2D, cartridge 1has a substantially planar housing fabricated, for example, from amoldable biocompatible material. The housing may be fabricated from anymaterial, however, transparent and/or translucent glass or polymers arepreferred. Preferred polymers include, for example, polystyrene,polycarbonate, acrylic, polyester, optical grade polymers, or anyplastic such that the optical cell is substantially transparent. Thehousing contains at least one fluid inlet port 4, at least one opticalcell 6, and at least one conduit 8 having a fluid contacting surface forproviding fluid flow communication between the fluid inlet port 4 andoptical cell 6. The only requirements for the optical cell 6 are that itdefines a void capable of containing a sample to be tested and that aportion of the optical cell 6 is transparent to light. Cartridge 1 mayalso have at least one pump port 12 in fluid flow communication withfluid inlet port 4 and optical cell 6 for attaching the cartridge 1 to apump. The pump may then impart a negative pressure via pump port 12 topull the sample from fluid inlet port 4 to optical cell 6. A hemocytelysate is disposed on a first region 14 of the fluid contacting surfaceof conduit 8, so that when a sample is applied to fluid inlet port 4,the sample traverses region 14 and solubilizes or reconstitutes thehemocyte lysate into the sample as it moves toward optical cell 6. Thistype of cartridge 1 may be used for performing, for example, an endpointturbidometric or a kinetic turbidometric assay. In an embodiment, achromogenic substrate may also optionally be applied to the surface ofthe conduit 8 at first region 14 together with the hemocyte lysate. Thistype of cartridge 1 may be used for performing, for example, a kineticchromogenic assay.

[0056] In a preferred embodiment, as illustrated in FIGS. 2A-2D, asecond region 16 of the fluid contacting surface of conduit 8 is spacedapart from and downstream of first region 14. In this configuration,hemocyte lysate is disposed at first region 14 and a chromogenicsubstrate is disposed at second region 16, so that after the sample iscontacted with the hemocyte lysate in region 14, the sample-lysatemixture traverses conduit 8 and contacts the chromogenic substrate inregion 16. The sample-lysate-substrate mixture then traverses conduit 8to optical cell 6. This type of cartridge may be used for performing,for example, an endpoint chromogenic assay or a multi-step kineticchromogenic assay, as discussed in more detail below.

[0057] Depending upon the type of assay to be performed, a pre-selectedamount of an agent representative of a microbial contaminant, or“spike,” such as a bacterial endotoxin, is disposed on first region 14of the fluid contacting surface of one or more conduits 8.Alternatively, the spike may be disposed on a different region of theconduit 8.

[0058] It is contemplated that the cartridge 1 may have a variety ofdifferent configurations, demonstrated, for example, in FIGS. 3 and 4.FIG. 3A shows a cartridge 1 comprising two conduits 8 with each conduit8 having its own sample inlet port 4. FIG. 3B shows a cartridge 1comprising two conduits 8 with each conduit 8 sharing a common sampleinlet port 4. FIG. 4A shows a cartridge 1 comprising four separateconduits 8 with each conduit 8 having its own sample inlet port 4. FIG.4B shows a cartridge 1 comprising two pairs of conduits 8, with eachpair having its own sample inlet port 4.

[0059] The cartridges can be designed and used according to the typeand/or number of tests required. For example, a single sample may betested, for example, in duplicate or triplicate, for example, forresearch laboratory uses or for medical device and biopharmaceuticaltesting. Alternatively, two or more different samples may be testedindividually, for example, for dialysis facility testing of water anddialysate. The cartridge preferably is a single-use, disposablecartridge that is discarded after one use. The cartridge of theinvention can use approximately 20-100 fold less hemocyte lysate persample than is used in the conventional endpoint chromogenic or kineticchromogenic assays performed in multi-well plates, and thus provides aless costly and environmentally-friendlier test.

[0060] Once a particular assay format has been chosen, the cartridge maybe fabricated as discussed below.

[0061] Cartridge Fabrication

[0062] All the reagents and materials used to prepare the cartridgepreferably are free of the microbial contaminant for which the cartridgeultimately will be used to test.

[0063] It is contemplated that the cartridge may be fabricated with anyhemocyte lysate of interest. As used herein, the term, “hemocyte lysate”is understood to mean any lysate or a fraction or component thereof,produced by the lysis and/or membrane permeabilization of hemocytes, forexample, amebocytes and hemolymph cells, (i) extracted from a crustaceanor insect and/or (ii) cultured in vitro after extraction from the host.Hemocyte cellular material that has been extruded from hemolymph cellsby contact with a membrane permeabilization agent such as a Ca²⁺ionophore or the like (i.e., extruded other than by lysis) or otherwiseextracted without cellular lysis is also considered to be a hemocytelysate. A preferred hemocyte lysate is an amebocyte lysate prepared fromthe blood of a crustacean, for example, a horseshoe crab or Jonah crab.It is also contemplated that hemocyte lysate may include a cocktail ofone or more natural (e.g., purified) or synthetic components of theenzyme cascades shown in FIG. 1.

[0064] As used herein, the term “amebocyte lysate” is understood to meanany lysate or fraction or component thereof produced by the lysis,extrusion, or extraction of the cellular contents from amebocytesextracted from a crustacean, for example, a horseshoe crab. Theamebocyte lysate comprises at least one component of an enzymaticcascade (for example, as shown in FIG. 1) and/or produces a clot in thepresence of an endotoxin, for example, a Gram negative bacterialendotoxin and/or a glucan, for example, a (1→3)-β-D glucan, produced bya yeast or a mold. Preferred amebocyte lysates can be derived fromhorseshoe crabs, which include crabs belonging to the Limulus genus, forexample, Limulus polyphemus, the Tachypleus genus, for example,Tachypleus gigas, and Tachypleus tridentatus, and the Carcinoscorpiusgenus, for example, Carcinoscorpius rotundicauda.

[0065] Crude lysates may be produced using the procedure as originallydescribed in Levin et al. (1968) Thromb. Diath. Haemorrh. 19: 186, withmodification, or in Prior (1990) “Clinical Applications of the LimulusAmebocyte Lysate Test” CRC Press 28-36 and 159-166, and in U.S. Pat. No.4,322,217. Other lysates may include those, for example, described inU.S. Pat. Nos. 6,270,982 and 6,391,570.

[0066] Presently, LAL is employed as the amebocyte lysate of choice inmany bacterial endotoxin assays because of its sensitivity, specificity,and relative ease for avoiding interference by other components that maybe present in a sample. LAL, when combined with a sample containingbacterial endotoxin and optionally with certain LAL substrates, reactswith the endotoxin in the sample to produce a detectable product, suchas a gel, increase in turbidity, or a colored or light-emitting product,in the case of a synthetic chromogenic substrate. The product may bedetected, for example, either visually or by the use of an opticaldetector.

[0067] As shown in FIG. 1, the coagulation system of hemolymph, like themammalian blood coagulation system, comprises at least two coagulationcascades that include an endotoxin-mediated pathway (the Factor Cpathway) and a (1→3)-B-D glucan-mediated pathway (the Factor G pathway).See, for example, Morita et al. (1981) FEBS Lett. 129: 318-321 andIwanaga et al. (1986) J. Protein Chem. 5: 255-268.

[0068] The endotoxin-mediated activation of LAL is well understood andhas been thoroughly documented in the art. See, for example, Levin etal. (1968) supra; Nakamura et al. (1986) Eur. J. Biochem. 154: 511; Mutaet al. (1987) J. Biochem. 101: 1321; and Ho et al. (1993) Biochem. &Mol. Biol. INT. 29: 687. When bacterial endotoxin is contacted with LAL,the endotoxin initiates a series of enzymatic reactions, referred to inthe art as the Factor C pathway, that can involve three serine proteasezymogens called Factor C, Factor B, and pro-clotting enzyme (see, FIG.1). Briefly, upon exposure to endotoxin, the endotoxin-sensitive factor,Factor C, is activated. Activated Factor C thereafter hydrolyses andactivates Factor B, whereupon activated Factor B activates proclottingenzyme to produce clotting enzyme. The clotting enzyme thereafterhydrolyzes specific sites, for example, Arg¹⁸-Thr¹⁹ and Arg⁴⁶-Gly⁴⁷ ofcoagulogen, an invertebrate, fibrinogen-like clottable protein, toproduce a coagulin gel. See, for example, U.S. Pat. No. 5,605,806.

[0069] It is possible to produce an endotoxin-specific lysate byremoving Factor G activity from the lysate, such as the Factor Gdepleted lysates produced by the methods described in U.S. Pat. Nos.6,391,570 and 6,270,982. Also, it is contemplated that lysates may bedepleted of Factor G activity by the addition of certain inhibitors ormodulators of Factor G activity, for example, certain detergents,saccharides, polysaccharides, and other reagents described in U.S. Pat.Nos. 5,155,032; 5,179,006; 5,318,893; 5,474,984; 5,641,643; 6,270,982;and 6,341,570. An endotoxin-specific lysate is a lysate capable ofreacting with a bacterial endotoxin but in which the reactivity to(1→3)-B-D glucan has been depleted by at least 80%, more preferably byat least 90%, and more preferably by at least 95% relative to the crudelysate from which the endotoxin-specific lysate was prepared.

[0070] (1→3)-B-D glucans and other LAL reactive glucans, produced bymicroorganisms such as yeasts and molds, can also activate the clottingcascade of LAL, through a different enzymatic pathway, referred to inthe art as the Factor G pathway (see, FIG. 1). Factor G is a serineprotease zymogen that becomes activated by (1→3)-β-D glucan or other LALreactive glucans. Upon exposure to (1→3)-β-D glucan, for example, FactorG is activated to produce activated Factor G. Activated Factor Gthereafter converts the proclotting enzyme into clotting enzyme,whereupon the clotting enzyme converts coagulogen into coagulin.

[0071] As used herein, the term, “(1→3)-β-D glucan” is understood tomean any water soluble polysaccharide, disaccharide or derivativethereof that is (i) capable of inducing formation of a coagulin clot incrude Limulus amebocyte lysate, and (ii) contains at least two β-Dglucosides, connected by a (1→3)-β-D glycosidic linkage (see Formula I).It is contemplated that such a polysaccharide or derivative thereof, inaddition to containing a (1→3)-β-D glycosidic linkage, may also containglucoside moieties connected by a variety of other glycosidic linkages,for example, via a (1→4)-β-D glycosidic linkage and/or by a (1→6)-β-Dglycosidic linkage. It is contemplated that such (1→3)-β-D glucans maybe isolated from a variety of sources including, without limitation,plants, bacteria, yeast, algae, and fungi, or alternatively may besynthesized using conventional sugar chemistries.

[0072] It is possible to produce a (1→3)-B-D glucan specific lysate byproducing a lysate depleted of Factor C activity. As shown herein, it ispossible to produce a glucan-specific lysate by lysing amebocytes in thepresence of at least 0.15 M salt, more preferably 0.25 M salt, forexample, a salt containing a monovalent cation, such as sodium orpotassium ions. For example, the amebocytes are lysed in about 0.15 M toabout 6 M salt, for example, sodium chloride. Alternatively, theamebocytes are lysed in a solution containing from about 0.25 M to about4 M salt, or from about 1 M to about 2M salt. When using sodiumchloride, it appears that the amoeboctye preparation loses substantialFactor C activity when the amebocytes are lysed in a solution containing0.25 M sodium chloride. However, the concentration of other saltsnecessary to produce a comparable results may be determined by routinetitration experiments. For example, amebocytes may be lysed in differentconcentrations of salt and the resulting lysates examined for theirability to produce a coagulin gel in the presence of a Gram negativebacterial endotoxin. The concentration of salt may be chosen where theresulting lysate has lost a substantial amount of reactivity to thebacterial endotoxin. Other salts that may be used include, but are notlimited to, monovalent ionic salts, such as, potassium chloride,potassium acetate and sodium acetate. An exemplary method for producinga glucan specific lysate is described in Example 4. A glucan-specificlysate is a lysate capable of reacting with glycan, for example,(1→3)-β-D glucan, but in which reactivity to a bacterial endotoxin orlipopolysaccharide has been depleted by at least 80%, more preferably atleast 90%, and more preferably at least 95% relative to the crude lysatefrom which the glucan-specific lysate was prepared.

[0073] Methods for enhancing the sensitivity of hemocyte lysate forendotoxin, for example, may include, without limitation, aging the crudehemocyte lysate, adjusting pH, adjusting the concentration of divalentcations, adjusting the concentration of coagulogen, chloroformextraction, and the addition of serum albumin, biocompatible buffersand/or biological detergents.

[0074] As will be apparent to one of ordinary skill, divalent metalsalts, which are known to promote activation of the pro-clotting enzymeof hemocyte lysate, as well as buffers to avoid extremes of pH thatcould inactivate the clotting enzyme preferably are included in thelysate. Any of the buffers and salts that are understood in the art tobe compatible with the amebocyte lysate system may be used. Typicalformulation additives may include, without limitation, about 100-300 mMNaCl, about 10-100 mM divalent cations (e.g., Mg²⁺ or Ca²⁺),biocompatible buffers, e.g., Tris (tris(hydroxy)aminomethane), to give afinal pH of about 6.0 to about 8.0, and, if the lysate is to be freezedried, then sugars, e.g., mannitol or dextran. It is contemplated thatthe choice of appropriate formulation additives may also be determinedby routine experimentation.

[0075] Synthetic chromogenic substrates have been used to measure thelevel of endotoxin-activated pro-clotting enzyme in LAL prepared fromthe hemolymph of both Tachypleus tridentatus and Limulus polyphemushorseshoe crabs (Iwanaga et al. (1978) Hemostasis 7: 183-188). During anLAL assay that uses a chromogenic substrate, the pro-clotting enzyme (aserine protease) in the LAL is activated by endotoxin and cleaves thesubstrate's peptide chain on the carboxyl side of arginine so as torelease the chromogenic group from the substrate, thereby releasing amarker compound that can be easily detected by, for example,spectrophotometry. One advantage of using a synthetic chromogenicsubstrate in an LAL assay in place of a conventional LAL gelation testis that the amount of activated clotting enzyme can be quantified andcorrelated to endotoxin levels in the sample.

[0076] Any chromogenic substrate that is cleaved by the clotting enzymeof a hemocyte lysate may be used in the practice of the invention. U.S.Pat. No. 5,310,657, for example, describes an exemplary chromogenicsubstrate having the formula R₁-A₁-A₂-A₃-A4-B-R₂, where R₁ representshydrogen, a blocking aromatic hydrocarbon or an acyl group; A₁represents an L or D-amino acid selected from Ile, Val or Leu; A₂represents Glu or Asp; A₃ represents Ala or Cys; A₄ represents Arg; Brepresents a linkage selected from an ester and an amide; and R₂represents a chromogenic of fluorogenic group which is covalentlyattached to the C-carboxyl terminal of Arginine through the B linkage,the fluorogenic or chromogenic moiety being capable of being cleavedfrom the remainder of the chromogenic substrate to produce a chromogenor a fluorogen. An exemplary chromogenic substrate has the consensussequence acetate-Ile-Glu-Ala-Arg-pNA, where pNA represents apara-nitroaniline group. U.S. Pat. No. 4,188,264 describes a peptidesubstrate with a structure consisting of L-amino acids in the sequenceR₁-Gly-Arg-R₂ where R₁ represents an N-blocked amino acid and R₂ is agroup that can be released by enzymatic hydrolysis to yield a coloredcompound, HR₂. U.S. Pat. No. 4,510,241 discloses a chromogenic peptidesubstrate, which differs from the previous substrate in that the Glymoiety is replaced in the sequence by Ala or Cys. Alternatively, thechromogenic substrate may contain a fluorophore, for example,7-amino-4-methyl coumarin, 7-amino-4-trifluoromethyl coumarin, and4-methoxy-2-naphthalyamine.

[0077] Inhibition or enhancement of the assay occurs when substances inthe test sample interfere with the hemocyte lysate reaction. Inhibitionresults in a longer reaction time, indicating lower levels of microbialcontamination than may actually be present in the test sample.Enhancement results in shorter reaction time, indicating higher levelsof microbial contamination than may actually be present in the testsample. To verify the lack of inhibition or enhancement, an aliquot oftest sample (or a dilution of the test sample) is “spiked” with a knownamount of an agent representative of the microbial contaminant to bemeasured. It is recommended that the microbial contaminant spike resultsin a final microbial contaminant concentration in the sample equal tothe mid-point, on a log basis, between the microbial contaminantconcentration of the highest and lowest standards in the standard curve.For example, in an assay with a standard curve spanning from 50Endotoxin Units (EU)/mL to 0.005 EU/mL, samples should be spiked tocontain a final microbial contaminant concentration of 0.5 EU/mL. In anassay with a standard curve spanning from 1 EU/mL to 0.01 EU/mL, themicrobial contaminant spike should result in a final microbialcontaminant concentration of 0.1 EU/mL.

[0078] The spiked sample is assayed in parallel with the unspikedsample. The resulting microbial contaminant concentration in theunspiked sample and the microbial contaminant recovered in the spikedsample then are calculated. The microbial contaminant recovered shouldequal the known concentration of the spike within about 25%. If the testsample (or dilution) is found to inhibit or enhance the reaction, thesample may require further dilution until the inhibition or enhancementis overcome. Initially, one may want to screen for inhibition orenhancement by testing 10-fold dilutions of test sample. Once theapproximate non-inhibitory or non-enhancing dilution is determined, theexact dilution can be found by testing two-fold dilutions around thisdilution. The degree of inhibition or enhancement will be dependent uponthe concentration of the test sample. If several concentrations of thesame sample are to be assayed, it is necessary to establish performancecharacteristics for each concentration independently.

[0079] In fabricating the cartridge of the invention, it is helpful tocombine the amebocyte lysate and chromogenic substrate with at least oneresolubilizing agent, such as a sugar or salt, and at least oneanti-flaking agent, such as a polymer, prior to drying the lysate ontothe solid support.

[0080] The resolubilizing agent preferably stabilizes the lysate in thedried form and facilitates resolubilization of the reagents during theassay. Useful resolubilizing agents include, for example, mannitol,mannose, sorbitol, trehalose, maltose, dextrose, sucrose, and othermonosaccharides and disaccharides. The hemocyte lysate and chromogenicsubstrate preferably contain from about 0.01% (w/v) to about 20% (w/v),more preferably from about 0.1% (w/v) to about 1.0% (w/v) of theresolubilizing agent prior to drying.

[0081] The anti-flaking agent is an agent that prevents or reduces thelikelihood that the hemocyte lysate and/or chromogenic substrate becomesdisassociated from a solid support in the form of a dry flake. Theanti-flaking agent preferably also stabilizes the hemocyte lysate orchromogenic substrate in the dried form. Useful anti-flaking agentsinclude, for example, one or more polymers, including, for example,polyethylene glycol, polyvinyl pyrolidone, dextrans, mannitol, andproteins, for example, serum albumin. The lysate preferably containsfrom about 0.01% (w/v) to about 25% (w/v), more preferably from about0.1% (w/v) to about 1.0% (w/v) of anti-flaking agent prior to drying.

[0082] In addition, it has been found that certain polymers reduce theformation of air bubbles (e.g., frothing) when the hemocyte lysateand/or chromogenic substrate are resolubilized. Useful anti-frothingagents include polyvinyl alcohol and polypropylene glycol. In order toreduce frothing, the lysate and/or chromogenic substrate may containfrom about 0.01% (w/v) to about 10% (w/v), more preferably from about0.1% (w/v) to about 1.0% (w/v) anti-frothing agent prior to drying.

[0083] An exemplary fabrication process for the cartridge is describedwith reference to FIG. 5, in which FIG. 5A represents a bottom half 2 ofcartridge 1 and FIG. 5B represents a top half 3 of cartridge 1. Onceprepared, the two halves of the cartridge 1 are joined to one another byadhesive, solvent bonding, ultrasonic welding, snap fit joints, or thelike.

[0084] In FIG. 5A, the bottom half 2 of the cartridge 1 defines one halfof each conduit 8′ (each having a first region 14′ and a second region16′). During fabrication of the bottom half 2 of the cartridge 1,hemocyte lysate is applied to each first region 14′ and chromogenicsubstrate is applied to each second region 16′. In FIG. 5B, the top half3 of the cartridge 1 defines one half of each conduit 8″. Duringfabrication of top half 3 of the cartridge 1, an agent representative ofa microbial contaminant (i.e., a spike), for example, a preselectedamount of an endotoxin, is applied to region 14″. Once the reagents havebeen applied to the respective top 3 and bottom 2 halves of thecartridge 1, the cartridge halves 2 and 3 then are dried underconditions that preserve the activity of the hemocyte lysate and permitreconstitution of the hemocyte lysate to produce active lysate. In orderto preserve the activity of the reagents during drying, the cartridgehalves 2 and 3 are placed in an environment having a temperature fromabout 4° C. to about 40° C., more preferably, from about 10° C. to about35° C., more preferably, from about 15° C. to about 30° C., and arelative humidity from about 0% to about 30%, more preferably, fromabout 2% to about 20%, more preferably from about 4% to about 10%.Preferred drying conditions include a temperature of about 25° C. and arelative humidity of about 5%. An exemplary protocol for manufacturing acartridge of the invention is provided in Example 1.

[0085] In an alternative approach, the hemocyte lysate may be dried viafreeze drying under standard conditions, about −30° C. to about −40° C.under vacuum.

[0086] After drying, the two cartridge halves 2 and 3 are joined to oneanother to create an intact cartridge 1. FIG. 5C is a cross-sectionalview through Section A-A′ in which the two halves of the conduit (namely8′ and 8″) together create an intact conduit 8, wherein region 14′ ofthe bottom 8′ of each conduit contains immobilized hemocyte lysate 20and region 14″ of the top 8″ of one conduit contains immobilizedendotoxin 22. FIG. 5D is a cross-sectional view through Section B-B′ inwhich region 16′ of the bottom 8′ of each conduit contains immobilizedchromogenic substrate 24.

[0087]FIGS. 6A-6D are illustrations of two exemplary cartridges 1 of theinvention corresponding to FIGS. 6A-6B and FIGS. 6C-6D. In FIG. 6A, thecartridge 1 may have an alternative finger grip 5 as shown with thedashed line. FIGS. 6A and 6B illustrate that the optical cell 6 in thefirst cartridge 1 is substantially cylindrical in shape. In FIG. 6C, thecartridge 1 also has a similar finger grip 5 to that shown by the dashedline in FIG. 6A. FIGS. 6C and 6D illustrate that the optical cell 6 insecond cartridge 1 is more elongate in shape. The elongate shape permitsgreater depth and rise of fluid for greater optical pathlength andproportionally greater detection sensitivity. In addition, it iscontemplated that the top and bottom halves 2 and 3 of each cartridge 1may comprise one or more male (female) members and one or morereciprocal and interfitting female (male) members to stack theunassembled cartridge halves one on top of the other, as well as providemating alignment in the assembled state.

[0088] The dimensions of a particular cartridge 1 may vary dependingupon the number and/or type of assays to be performed. However, in oneembodiment, as shown schematically in FIG. 6A, for example, thecartridge 1 has a length of about 10.16 cm (4.00″), width of about 2.54cm (1.00″), and a height of about 0.476 cm (.188″). The bore of theconduit 8 running from the fluid inlet port 4 to the optical cell 6 isabout 0.127 cm (.050″), where the lysate is dried on a region 14 of theconduit 8 about 2.381 cm (.938″) from the fluid inlet port 4, and achromogenic substrate is dried on a region 16 of the conduit 8 about4.65 cm (1.831″) from the fluid inlet port 4. The optical cell 6 in thisembodiment is dimensioned to accommodate about 25 μL of sample.

[0089] Specimen Collection and Preparation

[0090] The cartridge may be used to determine the level of microbialcontamination in a fluid, for example, a fluid to be administeredlocally or systemically, for example, parenterally to a mammal, or abody fluid to be tested for infection, including, for example, blood,lymph, urine, serum, plasma, ascites fluid, lung aspirants, and thelike. In addition, the cartridge may be used to determine the level ormicrobial contamination in a water supply, for example, a supply ofdrinking water. In addition, the cartridge may be used to determine thelevel of microbial contamination in a food product, pharmaceutical, ormedical device.

[0091] In general, materials used to harvest, store, or otherwisecontact a sample to be tested, as well as test reagents, should be freeof microbial contamination, for example, should be pyrogen-free.Materials may be rendered pyrogen-free by, for example, heating at 250°C. for 30 minutes. Appropriate precautions should be taken to protectdepyrogenated materials from subsequent environmental contamination.

[0092] Representative Assays that can be Performed in the Cartridge

[0093] It is contemplated that a variety of hemocyte lysate assays maybe used in the cartridge of the invention, such as, for example, the endpoint turbidometric assay, the kinetic turbidometric assay, the endpointchromogenic assay, and the single-step kinetic assay. In addition, thecartridge of the invention may be used with the multi-step kineticassay, as described herein.

[0094] 1. End Point Turbidometric Assay

[0095] The end point turbidometric assay is described in Prior (1990)supra, pp. 28-34. Briefly, the end point turbidimetric assay includesthe steps of (i) solubilizing a hemocyte lysate with a sample to beanalyzed, (ii) incubating the resulting mixture at a temperature ofabout 0° to about 40° C., preferably about 25° to about 40° C., for apredetermine and (iii) measuring the increase in turbidity as a resultof coagulation, if any, using a conventional coagulometer, nepherometer,or spectrophotometer.

[0096] Referring to FIG. 2A, in order to perform an endpointturbidometric assay in a cartridge 1, a sample is moved, for example, toa first region 14 of the conduit 8 containing the hemocyte lysate, whereit is solubilized, for example, by cycling between forward and reversepump action. The sample-lysate mixture then is moved to optical cell 6for measurement of an optical property, for example, turbidity, using anoptical detector. Results from multiple assays, for example, two assayscan be averaged. The optical density of the sample-lysate mixture at acertain predetermined time point may then be interpolated onto apredetermined standard curve, for example, showing turbidity values onthe Y axis versus endotoxin concentration on the X axis, to give theconcentration of the microbial contaminant in the sample.

[0097] 2. Kinetic Turbidometric Assay

[0098] The kinetic turbidometric assay is described in Prior (1990)supra, pp. 28-34. Briefly, the kinetic turbidimetric assay includes thesteps of (i) solubilizing a hemocyte lysate with a sample to beanalyzed, (ii) incubating the resulting mixture at a temperature ofabout 0° to about 40° C., preferably about 25° to about 40° C., over apredetermined time range, and (iii) measuring a time required for eithera turbidity change caused by coagulation to reach a pre-selected valueor a ratio in change of the turbidity, using a conventionalcoagulometer, nepherometer, or spectrophotometer.

[0099] Referring to FIG. 2A, in order to perform a kinetic turbidometricassay in a cartridge 1, a sample is, for example, moved to a firstregion 14 of the conduit 8 containing the hemocyte lysate, where it issolubilized, for example, by cycling between forward and reverse pumpaction. The sample-lysate mixture then is moved to optical cell 6 formeasurement of an optical property, for example, turbidity, by measuringthe absorbance or transmittance properties of the sample-lysate mixtureusing an optical detector. The detector may determine how long it takesfor each optical property to exhibit, for example, a 5% drop in opticaltransmittance. Results from multiple assays, for example, two assays canbe averaged. The resulting values may then be interpolated onto apredetermined standard curve, for example, showing time for apreselected change in transmittance on the Y axis versus endotoxinconcentration on the X axis, to give the concentration of thecontaminant in the sample.

[0100] 3. Endpoint Chromogenic Assay

[0101] The endpoint chromogenic assay is described in Prior (1990)supra, pp. 28-34, and U.S. Pat. Nos. 4,301,245 and 4,717,658. Briefly,the endpoint chromogenic assay includes the steps of (i) solubilizing ahemocyte lysate preparation with a sample to be analyzed, (ii)incubating the resulting mixture at a temperature of about 0° C. toabout 40° C., preferably about 25° C. to about 40° C., for apredetermined time, (iii) contacting a test device containingchromogenic substrate with the incubated sample-lysate mixture, (iv)adding a reaction inhibitor, and (v) measuring, e.g., by colorimetricchange, a substance released from the synthetic substrate by proteaseactivity.

[0102] Referring to FIG. 2A, in order to perform an endpoint chromogenicassay in a cartridge 1, a sample is moved, for example, to a firstregion 14 of the conduit 8 containing the hemocyte lysate, where it issolubilized, for example, by cycling between forward and reverse pumpaction. Following a predetermined incubation period, the sample-lysatemixture then is moved, for example, by pump action to a second region 16of the conduit 8 containing the chromogenic substrate, where it issolubilized, for example, by cycling between forward and reverse pumpaction. The sample-lysate-substrate mixture then is moved to a thirdregion containing a reaction inhibitor. Afterwards, thesample-lysate-substrate mixture then is moved to optical cell 6 formeasurement of an optical property, for example, the absorbance ortransmittance properties of the sample by an optical detector. Theoptical property of the sample-lysate-substrate mixture at a certainpredetermined time point may then be interpolated onto a predeterminedstandard curve, for example, showing absorbance, optical density, ortransmittance on the Y axis versus endotoxin concentration on the Xaxis, to give the concentration of the microbial contaminant in thesample.

[0103] 4. Single-Step Kinetic Assay

[0104] A single-step kinetic assay, for example, a singlestep-chromogenic assay, is described in U.S. Pat. No. 5,310,657.Briefly, the kinetic chromogenic assay includes the steps of (i)simultaneously solubilizing a hemocyte lysate with a sample to beanalyzed and a chromogenic substrate, (ii) incubating the resultingmixture at a temperature of about 0° to about 40° C., preferably about25° to about 40° C., over a predetermined time range, and (iii)measuring a time required for a colorimetric change to reach apre-selected value or a ratio in change of the colorimetric readout,using a conventional spectrophotometer.

[0105] Referring to FIG. 2A, in order to perform a kinetic chromogenicassay in a cartridge 1, a sample is moved, for.example, by pump action,to a first region 14 of the conduit 8 containing both the hemocytelysate and chromogenic substrate, where it is solubilized, for example,by cycling between forward and reverse pump action. Thesample-lysate-substrate mixture then is moved to optical cell 6 formeasurement of an optical property for example, the absorbance ortransmittance properties of the sample by an optical detector. Thedetector may determine how long it takes for each optical property toexhibit, for example, a 5% drop in optical transmittance. Results frommultiple assays, for example, two assays can be averaged. The resultingvalues may then be interpolated onto a predetermined standard curve, forexample, showing the time for a preselected change in absorbance ortransmittance (as the case may be) on the Y axis versus endotoxinconcentration on the X axis, to give the concentration of thecontaminant in the sample.

[0106] Of the above methods, the endpoint chromogenic assay and thesingle-step kinetic chromogenic assays currently are considered the mostrapid, sensitive, and economic assays for the detection of microbialcontaminants, for example, endotoxin. However, both assays have theirlimitations. The endpoint chromogenic assay is rapid (about 15 minutes)but typically can only detect endotoxin concentrations in a range thatis limited to about one log range (for example, about 1 EU/mL to about0.1 EU/mL), with a sensitivity of about 0.1 EU/mL. Although thesingle-step kinetic chromogenic assay measures endotoxin concentrationsin a wider range of about 5 logs (for example, about 5 to about 0.05EU/mL) with a high sensitivity of about 0.05 EU/mL, this method can bequite slow to run (about 30 minutes). Furthermore, neither the endpointchromogenic assay nor the single-step kinetic chromogenic assay arereadily adaptable for in-field performance. The multi-step kinetic assayovercomes the limitations in the endpoint chromogenic assay and thekinetic chromogenic assay.

[0107] 6. Multi-step Kinetic Assay

[0108] As will be discussed in more detail, the cartridge may also beused to perform a multi-step kinetic assay. The various steps involvedin the multi-step kinetic assay are shown schematically in FIG. 7. Theassay is initiated by combining the sample to be tested with a volume ofa hemocyte lysate to produce a sample-lysate mixture. The mixture thenis incubated for a predetermined period of time. The sample-lysatemixture then is contacted with a substrate, for example, a chromogenicsubstrate, to produce a sample-lysate-substrate mixture. Thereafter, thetime in which a preselected change in an optical property (for example,a specific change in an absorbance value or a specific change in atransmission value) is measured. The presence and/or amount of microbialcontaminant may be then determined by interpolating the measured timeagainst a pre-calibrated standard curve, for example, a standard curveshowing the time to make a preselected change in optical property(absorbance or transmittance) on the Y axis versus endotoxinconcentration on the X axis.

[0109] The standard curve may be created, for example, by addingincreasing amounts of an agent, for example, an endotoxin, glucan, orother microbial cell wall component, in a blank sample, for example,pyrogen-free water. The time for which a preselected change in anoptical property, for example, a preselected increase in absorbance or apreselected decrease in transmittance, is determined for eachconcentration of the microbial cell wall component. The various timemeasurements to achieve a standard change in optical property then areplotted as a function of the microbial cell wall componentconcentration. In general, the concentration of endotoxin is inverselyproportional to the time necessary to achieve the standard change inoptical property. The standard curve can then be used to assess thepresence and/or amount of microbial contaminant in the sample ofinterest. The calculation of standard curves is provided in Example 2.

[0110] As will be apparent to one skilled in the art, the relativeamounts of hemocyte lysate and substrate can be adjusted to ensure thateffective amounts of these two components are present in thesample-lysate-substrate mixture at the end of the assay. The finalamount of hemocyte lysate protein in the assay is from about 1 μg toabout 500 μg, preferably about 50 μg. The final amount of the substrate,for example, the chromogenic substrate in the assay is from about 1 μgto about 50 μg, preferably about 6.5 μg. The determination of theconcentration and composition of the substrate, for example, thechromogenic substrate, is considered to be within the level of skill inthe art.

[0111] The final volume of the resulting sample-lysate-substrate mixturecan be based on the requirements of the optical detector used to measurethe change in optical property of the sample. The ratio of volumesbetween the sample, lysate, and substrate can be readily established bythose of ordinary skill in the art. Depending on the relative volumes ofthe sample, lysate, and substrate in the sample-lysate-substratemixture, the concentration of the other components of the assay can beadjusted to maintain the final concentrations in the operable range, asdescribed herein.

[0112] Referring to FIG. 2A, to perform the multi-step kinetic assay ina cartridge 1 of the invention, a sample is first moved, for example, bypump action, to a first region 14 containing the hemocyte lysate, whereit is mixed and incubated for a predetermined period of time. Thesample-lysate mixture then is moved, for example, by pump action, to thesecond region 16 containing the substrate, for example, the chromogenicsubstrate, where it is solubilized. The sample-lysate-substrate mixturethen is moved to optical cell 6, for a measurement of an opticalproperty. The time intervals required for mixing and incubating stepsare preprogrammed for optimal sensitivity and microbial contaminantconcentration range.

[0113]FIGS. 8A and 8B show an exemplary cartridge and hand held opticaldetector useful in the practice of the invention. FIG. 8A shows thecartridge being introduced into the detector and FIG. 8B shows thecartridge inserted into the detector with the fluid inlet ports stillexposed to the user.

[0114]FIG. 9 is a graph showing the changes in optical properties thatcan be generated in a cartridge using a multi-step kinetic assay. Thedashed line represents changes in transmittance of the sample over time.The solid line represents changes in absorbance of the sample over time.Referring to FIGS. 2A and 9, optical properties were monitored after analiquot of sample was added to the fluid inlet port 4 at a time of 0seconds. After 60 seconds, the sample was drawn to region 14, where itwas mixed with hemocyte lysate disposed on region 14 for 60 seconds(represented by vertical zig zag lines from T=60 to T=120). Theamplitude of the mixing (length of the vertical lines) is determinedsuch that the sample is repeatedly moved over the entire region 14.Thereafter, the sample-lysate mixture was incubated from 120 to 480seconds at region 14 without further mixing. After 480 seconds, thesample was drawn to a chromogenic substrate at region 16 and thesample-lysate mixture combined with the chromogenic substrate from theperiod shown from 480 to 540 seconds (represented by vertical zig zaglines from T=480 to T=540). The amplitude of the mixing (length of thevertical lines) is determined such that the sample is repeatedly movedover the entire region 16. The resulting sample-lysate-substrate mixturethen was drawn to optical cell 6 and the optical properties (absorbanceand transmittance) measured for the period from 540 second to 1440seconds.

[0115] Using the initial absorbance or transmittance readings of themixture, the time required for the absorbance or transmittance to changeby an arbitrary amount (Reaction Time) is determined. The amount ofmicrobial contaminant in the sample then is determined by comparing theReaction Time for the sample against a predetermined standard curve.

[0116] A spiked sample is assayed in parallel with the unspiked sample.The microbial contaminant concentration in the unspiked sample and themicrobial contaminant recovered in the spiked sample can be compared todetermine the presence of interference, such as an inhibitor or anenhancer of the reaction, as previously described.

[0117] Although the multi-step assay may be performed in a cartridge ofthe type discussed above, it may be employed in a variety of otherformats, for example, within the well of a microtiter plate. Exemplaryassays performed in the well of a microtiter plate are discussed inExample 3. Multiple samples of various test fluids, as well as spikedsamples and the series of control samples making up a standard curve,may be placed in the wells of the microplate. Fixed amounts of hemocytelysate and then substrate are added to each of the wells, preferablyusing an automated system, such as a robot, and the plate processed by amicroplate reader, which can be programmed to sequentially read theabsorbance of each well in a repetitive fashion.

[0118] In addition, it is contemplated that the cartridges,glucan-specific assays, and the multi-step kinetic assays can be used todetect the presence and/or amount of a ligand of interest in a testsample. For example, by adapting the assay format as appropriate, it ispossible to detect the presence and/or amount of any ligand of interest,for example, a drug, toxin, protein, metabolite, in a sample. Anexemplary ligand assay performed in the well of a microtiter plate isdiscussed in Example 7. By way of example, a binder for a ligand ofinterest, for example, an antibody or antigen binding fragment thereof,is immobilized on the surface of a solid support, for example, in acartridge or a microplate. The binder is then pre-loaded or pre-boundwith a complex comprising the ligand of interest coupled or bound tolipopolysaccharide or glucan. Methods for conjugating glucan orlipopolysaccharide to a ligand are well known in the art. For example,Boutonnier et al. (2001) INFECT. IMMUN. 69:3488-3493, describe methodsfor conjugating lipopolysaccharide to tetanus toxoid (see also, Konaduet al. (1994) INFECT. IMMUN. 62:5048-5054; Kenne et al. (1982)CARBOHYDR. RES. 100:341-349). When a sample containing the ligand ofinterest is combined with the immobilized binder, the lipopolysaccharideor glucan-labeled ligand is displaced. The amount of displaced ligandcan then be quantified by the extent of the lipopolysaccharide or glucaninitiated reaction of a LAL preparation.

[0119] Using these principles, it is possible to create a cartridge fordetermining the presence and/or amount of a ligand of interest in asample. In this type of format, the binder for ligand is immobilized ona surface of the conduit downstream of the sample inlet port andupstream of the optical cell. The binder for ligand is then preloadedwith a complex comprising the ligand of interest coupled or conjugatedto, for example, lipopolysaccharide or glucan. A hemocyte lysate (forexample, a Factor G-specific lysate for detecting displaced glucan, or aFactor C-specific lysate for detecting displaced lipopolysaccharide) isimmobilized on a surface of the conduit downstream of the immobilizedbinder for ligand. When the sample of interest is applied to the sampleinlet port, the sample passes the binder for ligand. To the extent thatthe sample contains the ligand, the ligand displaces the complex fromthe immobilized binder. The amount of displaced ligand can be measuredby measuring a change in an optical property in the hemocyte lysate.This change in optical property can then be correlated with the amountof the ligand of interest in the sample.

EXAMPLES

[0120] Practice of the invention will be more fully understood from thefollowing examples, which are presented herein for illustrative purposesonly, and should not be construed as limiting the invention in any way.

Example 1

[0121] Preparation of the Chromogenic Assay Cartridge

[0122] An exemplary cartridge shown in FIGS. 2 was prepared as follows.Referring to FIG. 5A, the LAL and chromogenic substrates were applied toregions 14′ and 16′, respectively, of conduit 8′ of the bottom half 2 ofthe cartridge 1 using a Hamilton Microlab 540B Dispenser (HamiltonCompany, Reno, Nev.). Briefly, 4-5.0 μL of 10 mg/mL Endosafe LAL(Charles River Endosafe, Charleston, S.C.) containing 1% mannitol(Osmitrol, Baxter, Deerfield, Ill.) and 0.1% dextran (MW 10-100K,Sigma-Aldrich, St. Louis, Mo.), was applied to regions 14′. 4-5.0 μL of(1.3 mg/mL) chromogenic substrate Ile-Glu-Ala-Arg-pNA Chromogenix S-2423(Instrumentation Laboratories, Milan, Italy) containing 1% polyvinylalcohol (PVA) (MW 7K-30K, Sigma-Aldrich, St. Louis, Mo.), was applied toregions 16′. The bottom half 2 of the cartridge 1 was dried under acontrolled temperature of 25° C. +/−2° C. and a humidity of 5% +/−5% ina Lunaire Environmental Steady State & Stability Test Chamber (LunaireEnvironmental, Williamsport, Pa.) in a Puregas HF200 Heatless Dryer (MTIPuregas, Denver, Colo.) for 1 hour. Temperature and humidity wascontrolled by a Watlow Series 96 1/16 DIN Temperature Controller (WatlowElectric Manufacturing Company, St. Louis, Mo.).

[0123] Referring to FIG. 5B, 5μl Endosafe CSE endotoxin (Charles RiverEndosafe, Charleston, S.C.) (“spike”) was applied to region 14″ of theconduit 8″ of the top half 3 of the cartridge 1. The top half 3 of thecartridge 1 was dried under a controlled temperature of 25° C. +/−2° C.and a humidity of 5% +/−5% for one hour, as described above.

[0124] Following fabrication, the two halves 2 and 3 were assembled suchthat regions 14′ and 14″ were aligned one on top of the other, and theedges of the cartridge halves 2 and 3 ultrasonically sealed using aDukane Model 210 Ultrasonic Sealer (Dukane Corporation, St. Charles,Ill.) under the control of a Dukane Dynamic Process Controller (DukaneCorporation, St. Charles, Ill.).

[0125] The resulting cartridge 1 was labeled to identify the lot numberof the cartridge, in order to later identify the standard curves used toquantify the microbial contaminant in the sample. The sealed, labeledcartridge 1 then was placed into a laminated foil pouch along with adesiccant such as silica gel or molecular sieve. The foil pouch waspurged with nitrogen gas and then sealed with a PAC Model PV-G VacuumImpulse Heat Sealer (Packaging Aids Corporation, San Rafael, Calif.).

Example 2

[0126] Cartridge-based Assays

[0127] This example demonstrates that a cartridge of the invention canbe used to measure the amount of a microbial contaminant by an endpointchromogenic assay, kinetic chromogenic assay and a multi-stepchromogenic assay.

[0128]FIG. 8 shows how the cartridge of the invention may be used with aportable hand-held optical detector. FIG. 8A shows the cartridge in theprocess of being inserted into the optical detector. FIG. 8B shows thecartridge inserted fully into the optical detector, however, the fluidinlet ports of the cartridge are still accessible to the user. Thisconfiguration permits the user to apply one or more samples of interestto the exposed fluid inlet ports even though the optical cells of thecartridge are located in place within the optical detector.

[0129] (I) Endpoint Chromogenic Assay in a Cartridge

[0130] A cartridge was prepared essentially as described in Example 1,with the exception that no spikes were added to the conduits. Samples ofendotoxin standards of 1.0 EU/mL, 0.5 EU/mL, 0.25 EU/mL, and 0.125 EU/mLwere prepared and 25 μL of each were pipetted into one of four cartridgefluid inlet ports. The portable optical detector maintained atemperature of 37° C. for the duration of the test. The portable opticaldetector was programmed to perform a series of steps. The portableoptical detector first drew each endotoxin sample into the region of aconduit that contained dried LAL, and mixed and incubate the sample withthe LAL for 120 seconds (T=60 to T=180). The portable optical detectorthen drew the endotoxin sample-LAL mixture to the region in the conduitcontaining dried chromogenic substrate, and mixed the sample-LAL mixturewith the substrate for 5 seconds. The portable optical detector thendrew the sample-LAL-substrate mixture to the optical cell. The portableoptical detector then recorded absorbance data from each of the fourchannels. After about 10 minutes (about 780 seconds), the test was endedby reading the last absorbance (optical density) value. The absorbancevalue curves for each endotoxin sample are shown in FIG. 10A. Theabsorbance values generated at 780 seconds were recorded and plotted asa function of endotoxin concentration (FIG. 10B) to give a standardcurve. This standard curve can be used to determine the concentration ofendotoxin in a sample of interest, when the sample is treated in thesame manner as the standards.

[0131] (II) Single-Step Kinetic Chromogenic Assay in a Cartridge

[0132] A cartridge was prepared essentially as described in Example 1,with the exceptions that the chromogenic substrate was applied to thesame region as the LAL and that no spikes were added to the conduits.Samples of endotoxin standards of 5.0 EU/mL, 0.5 EU/mL, and 0.05 EU/mLwere prepared, and 25 μL of the 5 EU/mL standard was pipetted into twofluid inlet ports of the cartridge. The portable optical detectormaintained a temperature of 37° C. for the duration of the test. Theportable optical detector was programmed to perform a series of steps.The portable optical detector first drew each endotoxin sample into theregion of a conduit that contained both dried LAL and chromogenicsubstrate, and mixed and incubated the sample with the LAL and substratefor 30 seconds. The portable optical detector then drew thesample-LAL-substrate mixture to the optical cell. The portable opticaldetector began recording absorbance for both samples. The assay wasrepeated for the 0.5 EU/mL and 0.05 EU/mL standards.

[0133] The plots of recorded data for each endotoxin standard are shownin FIGS. 11A, 11B, and 11C. The time taken for the optical density ofeach endotoxin standard-LAL-substrate mixture to reach an opticaldensity of 0.05 was recorded as the onset time for each standard. A plotof the log of the endotoxin concentration (X axis) vs. the log of theonset times (Y axis) provides a kinetic standard curve (FIG. 11D). Thisstandard curve can be used to determine the concentration of endotoxinin a sample of interest when the sample is treated in the same manner asthe standards.

[0134] (III) Multi-step Kinetic Assay in a Cartridge

[0135] A cartridge was prepared essentially as described in Example 1,with the exception that no spikes were added to the conduits. Samples ofendotoxin standards of 1.0 EU/mL, 0.5 EU/mL, 0.25 EU/mL, and 0.125 EU/mLwere prepared and 25 μL of each were pipetted into one of four cartridgefluid inlet ports. The portable optical detector maintained atemperature of 37° C. for the duration of the test. The portabledetector was programmed to perform a series of steps. The portableoptical detector first drew each endotoxin sample into the region of aconduit that contained dried LAL, and mixed and incubate the sample withthe LAL for 240 seconds. The portable optical detector then drew theendotoxin sample-LAL mixture to the region in the conduit containingdried chromogenic substrate and mixed the sample-LAL mixture with thesubstrate for 20 seconds. The portable optical detector then drew thesample-LAL-substrate mixture to the optical cell. The portable opticaldetector began recording absorbance data from each of the four channels.The time taken for the optical density of each endotoxinstandard-LAL-substrate mixture to reach an optical density of 0.05 wasrecorded as the onset time for each standard (see, Table 1). A plot ofthe log of the endotoxin concentrations (X axis) versus the log of theonset times (Y axis) provides a kinetic standard curve (FIG. 12). Thisstandard curve can be used to determine the concentration of endotoxinin a sample when the sample is treated in the same manner as thestandards. TABLE 1 Endotoxin Concentration, EU/mL 5.0. 0.5 0.05 0 OnsetTime or Reaction Time 30 166 222 300 (seconds)

Example 3

[0136] Microplate-based Assays

[0137] This example demonstrates that a multi-well microtiter plate canbe fabricated so that the amount of a microbial contaminant (bacterialendotoxin in this example) can be measured by an endpoint chromogenicassay, a single-step kinetic assay and a multi-step assay.

[0138] (I) Single-step Kinetic Assay on a Microplate

[0139] A single-step kinetic chromogenic assay was performed as follows.Briefly, 50 μL of a control microbial contaminant of interest (e.g., 5EU/mL, 0.5 EU/mL, or 0.05 EU/mL of Endosafe Control Standard Endotoxin(CSE), Charles River Endosafe, Charleston, S.C.), was added to one ormore wells of a 96 well plate. 50 μL of 5 mg/mL Endosafe LAL (CharlesRiver Endosafe, Charleston, S.C.) and 5 μL of 1.3 mg/mL ChromogenixS-2423 chromogenic substrate (Instrumentation Laboratories, Milan,Italy) were added to each well and incubated at 37° C. for 32 minutes.The optical density of the mixture in each well was monitored by aspectrophotometer, such as a Sunrise micro plate reader (Tecan, ResearchTriangle Park, N.C.). The time taken for each standard to change 0.1absorbance units was determined (“onset time”). The results aresummarized below in Table 2. A plot of the log of the endotoxinconcentrations (X axis) versus the log of the onset times (Y axis)provides a kinetic standard curve (FIG. 13). This standard curve may beused to measure the concentration of endotoxin in a sample of interest,when the sample is treated in the same manner as the standards. TABLE 2Time to Onset OD/ Mean Time to Concentration, Max Reaction Time ReachOnset OD Standard (RT) Calculated Standard EU/mL (seconds) (seconds)Deviation CV % Value STD1 5.0 229.4/221.4 225.4 4.0/4.0 1.77 >4.9853STD2 0.5 472.2/465.0 468.6 3.59/3.59 0.77 0.5029 STD3 0.05 982.5/977.1979.8 2.68/2.68 0.27 <0.0499 CTRL1 0 >990.0/>990.0 >990.0 0.00/0.00 0.00<0.0500

[0140] (II) Endpoint Assay on a Microplate

[0141] An endpoint chromogenic assay using the reagents in Example 3(I)was performed as follows. Briefly, 50 μL of a control microbialcontaminant of interest (e.g., endotoxin) at a concentration of e.g., 5EU/mL, 0.5 EU/mL, or 0.05 EU/mL, was added to one or more wells of a96-well plate. 50 )L of hemocyte lysate (5 mg/mL) was added to one ormore wells containing the standard and incubated at 37° C. for 5minutes. 5 μL of chromogenic substrate (1.3 mg/mL) was added to one ormore wells and incubated at 37° C. for 5 minutes. The reaction then wasstopped by the addition of 100 μL of 50% acetic aid to each well. Theoptical density of the mixture in each well was measured at 405 nm by aspectrophotometer, such as a Sunrise micro plate reader (Tecan, ResearchTriangle Park, N.C.). The absorbance of each sample at 780 seconds(shown in Table 3) was recorded. The absorbance of each sample (Y axis)was plotted versus the endotoxin concentration (X axis) to provide astandard curve (shown in FIG. 14). This standard curve may be used tomeasure the concentration of endotoxin in a sample of interest, when thesample is treated in the same manner as the standards. TABLE 3Concentration, Optical Density Mean Optical Standard (OD) CalculatedStandard EU/mL (OD) Density Deviation CV % Value STD1 1.2 0.7505/0.81050.7805 0.03/0.03 3.84 1.1876 STD2 0.6 0.4135/0.3825 0.3980 0.02/0.023.89 0.6319 STD3 0.3 0.1655/0.1615 0.1635 0.00/0.00 1.22 0.2912 STD40.15 0.0655/0.0525 0.0590 0.01/0.01 11.02 0.1393 CTRL1 0  0.0115/−0.0115 0.0000 0.01/0.01 0.00 <0.1500

[0142] (III) Multi-step Kinetic Assay on a Microplate

[0143] A multi-step kinetic chromogenic assay using the same reagents asin Example 3(I) was performed as follows. Briefly, 50 μL of a controlmicrobial contaminant of interest, for example, endotoxin, at thefollowing concentrations of 5 EU/mL, 0.5 EU/mL, or 0.05 EU/mL, was addedto one or more wells of a 96-well plate. 50 μL of hemocyte lysate (5mg/mL) was added one or more wells and incubated at 37° C. for 3minutes. 5 μL of chromogenic substrate (1.3 mg/mL) was added to one ormore wells containing the standard and incubated at 37° C. for 16.5minutes. The optical density of the mixture in each well was monitoredby a spectrophotometer, such as a Sunrise micro plate reader (Tecan,Research Triangle Park, N.C.). The time taken for each microbialcontaminant standard and/or sample to change 0.1 absorbance units wasdetermined (“onset time”). The results are summarized in Table 4. A plotof the log of the endotoxin concentrations (X axis) versus the log ofthe onset times (Y axis) provides a kinetic standard curve (FIG. 15).This standard curve may be used to measure the concentration ofendotoxin in a sample of interest, when the sample is treated in thesame manner as the standards. TABLE 4 Time to Onset OD/ Mean Time toConcentration, Max Reaction Time Reach Onset OD Standard (RT) CalculatedStandard EU/mL (seconds) (seconds) Deviation CV % Value STD1 5.0172.1/166.4 169.3 2.84/2.84 1.68 >5.1505 STD2 0.5 393.8/387.7 390.73.03/3.03 0.78 0.4712 STD3 0.05 851.7/843.3 847.5 4.17/4.17 0.49 <0.0515CTRL1 0 >990.0/>990.0 >990.0 0.00/0.00 0.00 <0.0500

Example 4

[0144] Preparation and Testing of Glucan-Specific LAL

[0145] As shown in FIG. 1, crude LAL reacts with both endotoxin(lipopolysaccharide) and glucan, so that cell wall material from bothGram negative bacteria and yeast/mold cells activate the coagulationcascade. In the case of Gram negative bacteria, coagulation is mediatedthrough the Factor C cascade. In the case of yeast and mold, coagulationis mediated through the Factor G cascade. In order to determine which ofthe two contaminants is present in a sample, or what proportion of eachmight comprise a mixed contamination, LAL was produced under conditionsto render it specific for glucan, as described below.

[0146] Amebocyte blood was mixed 6:1 with 0.05% Tween 20 (Sigma, St.Louis, Mo.), 150 mM NaCl and centrifuged in a Sorval model RC-3Bcentrifuge at 3,000 rpm for 5 minutes. The pelleted cells were washedwith 0.01% Tween 20, 150 mM NaCl and centrifuged at 3,000 rpm for 5minutes. The pelleted, washed cells were divided into three aliquots,resuspended in either lipopolysaccharide-free water,lipopolysaccharide-free 1 M NaCl, or lipopolysaccharide-free 2 M NaCl.The cells in each pellet were lysed by sonication for 1-2 minutes. Celldebris was removed by centrifugation at 4,000 rpm for 10 minutes, andthe resulting supernatant was harvested and used directly in coagulationexperiments.

[0147]FIG. 16 provides a graphical representation of kinetic coagulationreactions using various concentrations of lipopolysaccharide (FIG. 16A)or glucan (FIG. 16B) in a microtiter plate multi-step kinetic assay,such as that described in Example 3 (III) with either standard LAL (rowA of each figure) or glucan-specific LAL (rows B and C in each figure).

[0148]FIG. 16A illustrates the reactivity of the lysates withlipopolysaccharide (Charles River Endosafe) serially diluted 1:10 fromcolumn 1 to column 5. The lipopolysaccharide concentrations in columns 1through 5 are: 10 ng/ml, 1 ng/ml, 100 pg/ml, 10 pg/ml, and 0,respectively.

[0149]FIG. 16B illustrates the reactivity of the lysates with glucan(Charles River Endosafe, Charleston, S.C.) serially diluted 1:10 fromcolumn 1 to column 5. Glucan concentrations from column 1 through 5 are:100 μg/ml, 10 μg/ml, 1 μg/ml, 100 ng/ml, and 0, respectively.

[0150] In each figure, row A shows the reactivity with standard LALprepared from cells lysed in pyrogen-free water (i.e., reactive withboth glucan and lipopolysaccharide). In each figure, row B isglucan-specific LAL produced by lysing cells in 1 M NaCl. In eachfigure, row C is glucan-specific LAL produced by lysing cells in 2MNaCl. Each graph represents the change in optical density or absorbance,as shown on the Y axis, over time, as shown on the X axis. For example,in FIG. 16A, the graph shown in row A, column 1, shows the change inabsorption over time when 10 ng/ml lipopolysaccharide is added tostandard LAL.

[0151] The results demonstrate that when lipopolysaccharide is added tostandard lysate, the lipopolysaccharide activates the lysate to producean increase in absorbance (see, FIG. 16A, row A). However, the rate ofabsorbance change decreases as less lipopolysaccharide is added to eachsample. The results demonstrate, however, that there is substantially nochange in absorbance as lipopolysaccharide is added to eachglucan-specific lysate (see, FIG. 16A, rows A and B).

[0152] The results also demonstrate that when glucan is added tostandard lysate, the glucan (like the lipopolysaccharide) activates thelysate to produce an increase in absorbance (see, FIG. 16B, row A).However, the rate of absorbance change decreases as less glucan is addedto each sample. In contrast to the situation when lipopolysaccharide wasadded, the glucan activates each glucan-specific lysate to produce anincrease in absorbance over time (see, FIG. 16B, rows B and C).

[0153] These results demonstrate that it is possible to produce aglucan-specific lysate using the protocol described herein.

Example 5

[0154] Testing of Glucan-Specific LAL in a Cartridge-based Multi-StepAssay

[0155] Glucan-specific LAL was prepared by lysing amebocytes in 2M NaClas described in Example 4 and tested in a cartridge-based multi-stepkinetic assay, such as that described in Example 2(111).

[0156] Samples containing glucan at 100 μg/ml, 10 μg/ml, 1 μg/ml, 100ng/ml, and 0, were incubated with the glucan-specific lysate for 4minutes. The resulting mixture was mixed with a chromogenic substrate,acetate-Ile-Glu-Ala-Arg-pNA and the change in absorbance at 405 nm wasmeasured over time. The time required to reach an onset optical densityof 0.05 was collected (see, Table 5) and the log of the glucanconcentration was plotted versus the log of the time to reach onset O.D.(see, FIG. 17). TABLE 5 Glucan Standard Onset Time Calculated μg/ml(μg/ml) (seconds) glucan activity 100 207 80.5 10 400 15.4 1 1300 0.800.1 >1800 <0.36 Negative control >1800 <0.36

[0157] The data show that it is possible to produce a standard curve ofglucan concentration using a cartridge-based multi-step assay. Thestandard curve can then be used to determine the concentration of glucanin a sample of interest, when the sample is treated in the same manneras the standards.

Example 6

[0158] Means of Reading an LAL Reaction Using Fluorescent Substrates

[0159] A cartridge-based multi-step kinetic LAL assay, such as thatdescribed in Example 2(III), was performed using the fluorogenicsubstrate: Glu-Gly-Arg-AMC (Enzyme Systems Products, Livermore, Calif.).The cartridge was modified to include a long-pass filter placed betweenthe sample and the light sensor such that light having the excitationwavelength (390 nm) was blocked but yet the emissions at a wavelength of460+/−25 nm were able to pass through and be detected by the sensor.

[0160] Using the device, fluorescence data (expressed as RelativeFluorescence Units) were collected from samples containing 10 EU(Endotoxin units), 1 EU, and 0.1 EU. The changes in RelativeFluorescence Units over time are presented in FIG. 18. The resultsdemonstrate that the multi-step kinetic assays can measure the amount ofendotoxin in sample using a fluorescent substrate.

Example 7

[0161] Measurement of Lipopolysaccharide-labeled Ligand Using anImmobilized Antibody

[0162] The microtiter plate multi-step kinetic LAL assay, such as thatdescribed in Example 3(111) can also be used to measure theconcentration of a ligand. Briefly, an antibody that binds a ligand ofinterest is immobilized onto the surface of a well of a microtiterplate. Then, the ligand binding sites of the antibody are preloaded witha complex comprising the ligand coupled to lipopolysaccharide. When asample containing the ligand is exposed to the immobilized antibody, thelipopolysaccharide-labeled ligand is displaced and quantified byreaction with LAL. In this example, the ligand detected was fluorescein.An anti-fluorescein antibody was immobilized onto the surface of a welland was pre-loaded with fluorescein-labeled lipopolysaccharide. Whensamples containing fluorescein-labeled antibody were exposed to theimmobilized antibody, the fluorescein-lipopolysaccharide conjugate wasreleased from the immobilized antibody and measured by reactivity withLAL.

[0163] Briefly, rabbit anti-fluorescein antibody (Virostat, PortlandMe.) was diluted 1/4000 in CAPS buffer, pH 10.2 (Sigma, St. Louis, Mo.).25 μl of the diluted antibody was added to the wells of a high bindingplate (Coming 25801) and incubated for 1 hour at 37° C. The plate waswashed 4×100 μl per well with TTBS (0.1% Tween, 100 mM Tris bufferedsaline) using a multipipettor. The wells were blocked by adding 150 μlper well of gelatin diluent and stored at 4° C. overnight. The wellsthen were washed with 3×100 μl per well with TTBS. Lipopolysaccharide(LPS)-fluorescein (FITC) conjugate (List Biological Laboratory,Campbell, Calif.) was diluted from 1 μg/ml to 10 pg/ml using 10-folddilutions in 0.1 M Tris. 0.1 M Tris was used as a control. 75 μl ofdiluted LPS/FITC conjugate was added per well and incubated for 30minutes at 37° C. The wells then were washed with 3×100 μl per well with0.1 M Tris.

[0164] Fluorescein-labeled goat anti-chicken antibody (Southern Biotech.Associates Inc 6100-02, Birmingham, Ala.) was diluted 1/50, 1/150, and1/450 in 0.1 M Tris. 0.1 M Tris was used as a control. 75 μl offluorescein-labeled goat anti-chicken antibody was added per well andincubated for 30 minutes at 37° C. 50 μl was removed from each well andtransferred to a clean 96-well plate (Falcon, 353072, Becton Dickenson,Franklin Lakes, N.J.). 50 μl Endochrome K (Charles River Endosafe),which is a 1:1 mixture of LAL substrate and LAL lysate, was added toeach well. The plate was read at time intervals (e.g., kinetically) at405 nm at 37° C. for 60-90 minutes (Min OD: 0, Max OD: 0.8, Onset OD:0.1).

[0165] To find the proper concentration of LPS/FITC to be pre-bound tothe immobilized antibody, increasing concentrations were applied to theplate from 10 pg/mL to 1 μg/mL. Then dilutions of thefluorescein-labeled antibody ligand were exposed to the immobilizedantibody and the displaced LPS/FITC measured as EU equivalents. As shownin FIG. 19, at the 1 μg/mL level of LPS/FITC, an EU proportional to theligand was achieved (far left). Accordingly, by using this type offormat, it is possible to detect the presence and/or measure the amountof a ligand of interest in a test sample.

Equivalents

[0166] The invention may be embodied in other specific forms withoutdeparting from the spirit or essential characteristics thereof. Theforegoing embodiments are therefore to be considered in all respectsillustrative rather than limiting on the invention described herein.Scope of the invention is thus indicated by the appended claims ratherthan by the foregoing description, and all changes that come within themeaning and range of equivalency of the claims are intended to beembraced therein.

Incorporation by Reference

[0167] All publications and patent documents cited in this applicationare incorporated by reference in their entirety for all purposes to thesame extent as if the entire contents of each individual publication orpatent document was incorporated herein.

1. A cartridge for determining the presence or amount of a microbialcontaminant in a sample, the cartridge comprising: (i) a housingdefining a fluid inlet port, an optical cell, and a conduit having afluid contacting surface for providing fluid flow communication betweenthe fluid inlet port and the optical cell; and (ii) hemocyte lysatedisposed on a region of the fluid contacting surface of the conduit, sothat when a sample is applied to the fluid inlet port, the sampletraverses the region and solubilizes the hemocyte lysate duringtransport to the optical cell.
 2. The cartridge of claim 1, furthercomprising a chromogenic substrate disposed on a second region of thefluid contacting surface.
 3. The cartridge of claim 2, wherein thesecond region is downstream of the first region.
 4. The cartridge ofclaim 1, further comprising a preselected amount of an agentrepresentative of the microbial contaminant disposed on the fluidcontacting surface of the conduit.
 5. The cartridge of claim 4, whereinthe agent is disposed on the first region.
 6. The cartridge of claim 4,wherein the agent is a bacterial endotoxin or a (1→3)-β-D glucan.
 7. Acartridge for determining the presence or amount of a microbialcontaminant in a sample, the cartridge comprising: (i) a housingdefining a first fluid inlet port, a first optical cell, and a firstconduit having a fluid contacting surface for providing fluid flowcommunication between the first fluid inlet port and the first opticalcell, and a second fluid inlet port, a second optical cell, and a secondconduit having a fluid contacting surface for providing fluid flowcommunication between the second fluid inlet port and the second opticalcell; (ii) a first hemocyte lysate disposed on a first region of thefluid contacting surface of the first conduit, so that when a sample isapplied to the first fluid inlet port, the sample traverses the regionand solubilizes the first hemocyte lysate during transport to the firstoptical cell; and (iii) a second hemocyte lysate disposed on a firstregion of the fluid contacting surface of the second conduit, so thatwhen sample is applied to the second fluid inlet port, the sampletraverses the region and solubilizes the second hemocyte lysate duringtransport to the second optical cell.
 8. The cartridge of claim 7,further comprising a chromogenic substrate disposed on a second regionof the fluid contacting surface of the first conduit.
 9. The cartridgeof claim 8, wherein the second region is downstream of the first region.10. The cartridge of claim 8, further comprising a chromogenic substratedisposed on a second region of the fluid contacting surface of thesecond conduit.
 11. The cartridge of claim 10, wherein the second regionis downstream of the first region.
 12. The cartridge of claim 7, furthercomprising a preselected amount of an agent representative of amicrobial contaminant disposed on the fluid contacting surface of thefirst conduit.
 13. The cartridge of claim 12, wherein the agent isdisposed on the first region.
 14. The cartridge of claim 12, wherein theagent is a bacterial endotoxin or a (1→3)-β-D glucan.
 15. A method ofdetecting the presence of a microbial contaminant in a sample, themethod comprising the steps of: (a) introducing a sample into the sampleinlet port of the cartridge of claim 1; (b) permitting the sample tomove to the optical cell; and (c) measuring an optical property of thesample in the optical cell, wherein a change in the optical property isindicative of the presence of a microbial contaminant in the sample.16-17. (Cancelled)
 18. A method of determining the amount of a microbialcontaminant in a sample, the method comprising the steps of: (a)introducing a sample into the sample inlet port of the cartridge ofclaim 1; (b) permitting the sample to move to the optical cell; (c)measuring the time in which a preselected change occurs in an opticalproperty of the sample in the optical cell; and (d) comparing the timemeasuring in step (c) against a predetermined standard curve todetermine the amount of the microbial contaminant in the sample. 19-20.(Cancelled)
 21. A method of determining the presence of a microbialcontaminant in a sample, the method comprising the steps of: (a)contacting a sample with a hemocyte lysate comprising an activatableenzyme to produce a sample-lysate mixture, whereupon the enzyme becomesactivated if the microbial contaminant is present in the sample; (b)after step (a), contacting the sample-lysate mixture with a substratefor the enzyme to produce a sample-lysate-substrate mixture, such that,if the mixture contains activated enzyme, the activated enzyme producesa change in the substrate; (c) determining the time in which apreselected change occurs in an optical property of thesample-lysate-substrate mixture, wherein the change in optical propertyresults from a change in the substrate; and (d) comparing the timedetermined in step (c) against a predetermined standard curve todetermine whether the contaminant is present in the sample. 22-43.(Cancelled)
 44. A method of drying a hemocyte lysate onto a solidsupport, the method comprising the steps: (a) applying a volume of amixture comprising a hemocyte lysate, a resolubilizing agent and ananti-flaking agent to the surface of a solid support; and (b) drying themixture onto the solid support. 45-58. (Cancelled)
 59. A method ofpreparing an amebocyte lysate depleted of Factor C activity, the methodcomprising: (a) providing a preparation of amebocytes; and (b) lysingthe amebocytes in the presence of at least 0.15 M salt to provide anamebocyte lysate preparation depleted of Factor C activity. 60-69.(Cancelled)
 70. An amebocyte lysate substantially free of Factor Cactivity, wherein the lysate comprises at least about 0.25 M salt, andwherein the lysate is capable of reacting with glucan to produce acoagulin gel. 71-76. (Cancelled)
 77. A manufacture comprising a solidsupport having dried thereon a composition comprising a hemocyte lysateand (i) an anti-flaking agent, (ii) an anti-frothing agent, (iii) aresolubilizing agent, (iv) an anti-flaking agent and an anti-frothingagent, (v) an anti-flaking agent and a resolubilizing agent, (vi) ananti-frothing agent and a resolubilizing agent, or (vii) an anti-flakingagent, an anti-frothing agent and a resolubilizing agent. 78.(Cancelled)